The present application relates to amino acid sequences comprising epitopes of adenyl cyclase-haemolysin from Bordetella. Adenyl cyclase-haemolysin (AC-Hly) is one of the toxins participating in the Bordetella infectious syndrome. AC-Hly is a bifunctional protein having an adenyl cyclase activity and a haemolytic activity. It is secreted by the bacterium. Its structural gene has been cloned and sequenced (Glaser P. et al., 1988, Molec. Microb. 2, 19-20). It is the case that this protein is part of the family of toxins termed "RTX" for "repeats in toxins" and exhibits homologies with haemolysin from Escherichia coli and Actinobacillus pleuropneumoniae, and the leucotoxins from Pasteurella haemolytica and Actinobacillus actinomycetemcomitans. This protein, like PTX (pertussis toxin), is capable of penetrating into eucaryotic cells such as the macrophages, of being activated by calmodulin, of synthesizing large quantities of cAMP and of disrupting cellular functions (Coote J. 1992. FEMS Microbiol. Rev. 88:137-162).
The inventors have identified, within this sequence, various domains having the capacity to induce the formation of protective antibodies against an infection by Bordetella, in particular by B. pertussis and/or B. parapertussis and/or B. bronchiseptica.
The subject of the invention is therefore amino acid sequences capable of entering into the composition of immunogenic compositions or of protective vaccines against Bordetella infections as well as antibodies directed against these amino acid sequences, capable of being used for example in immunotherapy. Immunotherapy or serotherapy is especially applicable in children, where appropriate in infants infected with B. pertussis, B. parapertussis or B. bronchiseptica. The invention proposes applications in human medicine or in veterinary medicine.
The subject of the present invention is an amino acid sequence derived from the polypeptide sequence of adenyl cyclase-haemolysin (AC-Hly), characterized in that it is capable of inducing the formation of protective antibodies against an infection by B. pertussis and/or B. parapertussis and/or B. bronchiseptica, and in that it is chosen from the following chains:
a) a sequence comprising the chain of amino acids situated approximately between position 910, preferably 913, and the last C-terminal amino acid of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO: 2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronshiseptica (SEQ ID NO: 4), the said sequence comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983; PA1 b) a sequence comprising a chain of 6 to 500 amino acids comprising amino acids 385 to 400 of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO: 2), one in B. parapertussis or in B. bronshiseptica (SEQ ID NO: 4); PA1 c) a sequence comprising the chains of amino acids defined above in a) and b), the said chains being either contiguous, or combined via the chain of amino acids naturally present between the chains defined above in a) and b) within the AC-Hly from B. pertussis, B. parapertussis or B. bronshiseptica, or combined via an antigenic sequence derived from a protein different from AC-Hly, the amino acid sequence obtained having a three-dimensional conformation identical or analogous to that of the corresponding polypeptide sequence of AC-Hly from B. pertussis, B. parapertussis or B. bronchiseptica. PA1 immunizing an animal, for example a Balb/c mouse with a peptide comprising the sequence of amino acids 385 to 400 of AC-Hly from B. pertussis (SEQ ID NO:2), the immunization being, where appropriate, carried out by means of repeated administrations of the peptide; PA1 fusing the spleen cells of the immunized animal with myeloma cells to form a hybridoma; PA1 culturing the hybridoma under conditions allowing the production of antibodies; PA1 recovering the antibodies directed against the sequence of amino acids 385 to 400 of the AC-Hly from B. pertussis.
The expression "amino acid sequence derived from the polypeptide sequence of AC-Hly" is understood to mean, within the framework of the invention, a sequence whose amino acids are identical, by their nature or by their linkage to those of the polypeptide sequence of AC-Hly or are, for some of them, substituted, deleted or added, in a manner such that the immunological properties of AC-Hly are conserved. In particular, such a sequence, derived from the polypeptide sequence of AC-Hly is recognized by antibodies formed in a patient infected with Bordetella, especially with B. pertussis, B. paprapertussis or B. bronchiseptica.
It will be considered, within the framework of the present invention, that the structure or the conformation of AC-Hly is conserved, the amino acid sequence of the invention then having a structure identical or analogous to that of AC-Hly, when the said amino acid sequence is capable, after immunization of a patient or an animal, of inducing a protective immunity against Bordetella infections.
A sequence according to the invention can be obtained by proteolysis of AC-Hly purified from Bordetella. Preferably, this sequence is obtained by chemical synthesis or by genetic engineering techniques.
Thus, to produce an amino acid sequence according to the invention by genetic engineering, plasmide carrying fragments of the CyaA gene will be used.
By way of example, the chemical synthesis may be performed in automatic machines of the Applied Biosystem type. It is also possible to use the technique of Betsou F. et al., 1993, Infect. Immun., 61:3583-3589.
In this manner, it will be possible to prepare a peptide corresponding to the chain of amino acids 385 to 400, or even 385 to 500 of AC-Hly from B. pertussis or to the corresponding chain of AC-Hly from B. parapertussis or from B. bronchiseptica. The amino acid sequence (SEQ ID NO:2) as well as the nucleotide sequence (SEQ ID NO:1) of AC-Hly from B. pertussis has been described in Glaser et al, 1998, Molec. Microb. 2-19-30 and is represented in FIG. 5. The amino acid sequence (SEQ ID NO:4) and the nucleotide sequence (SEQ ID NO:3) of AC-Hly from B. bronchiseptica is represented in FIG. 6.
The amino acid sequence of AC-Hly from B. parapertussis and the nucleotide sequence encoding AC-Hly can be obtained by conventional techniques from the DNA from a strain of B. parapertussis, for example the strain No. 1 deposited at the CNCM on Dec. 2, 1994 under the No. I-1498.
When an amino acid sequence according to the invention is produced by genetic engineering from the DNA of the CyaA genes from B. pertussis, B. parapertussis or B. bronchiseptica, and when it comprises the amino acid in position 983 of AC-Hly from B. pertussis (SEQ ID NO:2or a corresponding amino acid of AC-Hly from B. parapertussis or B. bronchiseptica (SEQ ID NO:4), care will be taken to ensure that this sequence is produced in a cellular host also expressing the CyaC gene from the strains identified above. The expression of the CyaC gene allows the modification by addition of a fatty acid necessary to conserve in the amino acid sequence comprising residue 983. The addition of a fatty acid may, by way of example, be a palmitoylation.
Strains which can be used in order to have access to CyaA and CyaC genes are for example B. pertussis HAV deposited at the CNCM on Oct. 19, 1994 under the No. I-1485, B. parapertussis I-1498 and B. bronchiseptica 973S deposited at the CNCM on May 12, 1989 under the No. I-858.
Moreover, the nucleotide sequences encoding AC-Hly from B. pertussis and from B. bronchiseptica are presented in FIGS. 5 and 6 (SEQ ID NO:1 and 3respectively.
The nucleotide sequence of the CyaC gene which activates the CyaA gene has been described in the publication of Barry E. M. et al. (Journal of Bacteriology, January 1991, p. 720-726)